IST-301 D-Seal – MicroPlate Seal Removal Tape

Overview

• Detection kits for Epstein-Barr Virus (EBV)
• Lyophilized format designed for room temperature shipping
• Available in TaqMan format for analysis

Norgen’s EBV TaqMan PCR Lyophilized Kit is designed for the detection of EBV specific DNA in a real-time PCR based on the use of TaqMan® technology. The lyophilized format is designed to ship the kit at ambient temperature.

Norgen’s EBV TaqMan Lyophilized Probe/Primer and Control Set is designed for the detection of EBV specific DNA in a real-time PCR based on the use of TaqMan® technology. The lyophilized format is designed to ship the kit at ambient temperature.

Details

Supporting Data

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Storage Conditions and Product Stability

• All kit components should be stored at -20°C upon arrival.
• Once reconstituted, repeated thawing and freezing (>2 times) of the Master Mix and Positive Control should be avoided, as this may affect the performance of the assay. If the reagents are to be used only intermittently, they should be frozen in aliquots.
• Each kit is provided with 4 tubes of 2X PCR Master Mix and each tube is enough to run 25 reactions. It is not necessary to reconstitute all Master Mix tubes at once. The Master Mix tubes can be reconstituted as and when needed.
• All kit components can be stored for 2 years after the date of production without showing any reduction in performance.

Component	Cat. TM41050L (100 preps)	Cat. TM41010L (100 preps)
MDx TaqMan 2X PCR Master Mix (Lyo)	4 x 350 μL	–
EBV Primer & Probe Mix (Lyo)	280 μL	280 μL
EBV Positive Control (Lyo)	150 μL	150 μL
Nuclease-Free Water (Negative Control)	3 x 1.25 mL	1 x 1.25 mL
Product Insert	1	1

Opentrons Thermocycler GEN2 Seals are adhesive-backed Ethylene Propylene Diene Monomer (EPDM) seals designed for use with the fully-automated Thermocycler Module GEN2. They come in a package of 10 seals. These seals need to be applied to the hood of the Thermocycler and can be reused up to 20 times. Opentrons Thermocycler GEN2 Seals need to be sterilized before each use.

Note, if you have a Thermocycler Module GEN1, please use the Thermocycler GEN1 Seals.

Opentrons Thermocycler GEN2 Seals are adhesive-backed Ethylene Propylene Diene Monomer (EPDM) seals designed for use with the fully-automated Thermocycler Module GEN2. They come in a package of 10 seals. These seals need to be applied to the hood of the Thermocycler and can be reused up to 20 times. Opentrons Thermocycler GEN2 Seals need to be sterilized before each use.

Note, if you have a Thermocycler Module GEN1, please use the Thermocycler GEN1 Seals.

Bisulfite seq is a well know technology to detect DNA methylation and several technologies such as WGBS, RRBS, MeDIP-Seq, and MSBS are used for whole genome DNA methylation analysis. DNA methylation is important for regulation of cell development, differentiation and gene expression in molecular biology, genetics and epigenetics. Most methylated cytosines are found at CpG sites, and 70-80% of cytosines are methylated. The number of CpG sites in human genome is around 28 million, which is less than 1% of the genome compared with 4.4% expected.

Whole genome bisulfite sequencing (WGBS) is the most effective method of DNA methylation analysis. The only limitation is the sequencing cost is very high because the whole genome is sequenced including all the non-methylated regions.

Reduced Representation Bisulfite Sequencing (RRBS) is the reduced representation of a smaller fraction of the methylated CpG sites. RRBS combines restriction enzyme digestion and bisulfite sequencing, and enriches the sequencing for methylated CpG sites. It is an efficient technology for estimate the whole genome methylation patterns at the single base level. Although this allows a higher coverage depth and reduces the sequencing cost, the limitation is only 10% of the methylated CpG sites are covered.

Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq) is another whole genome enrichment technique used for selection of methylated DNA. Using antibodies against 5-methylcytosine, methylated DNA is enriched from whole genomic DNA via immunoprecipitation. 5-methylcytosine antibodies are incubated with fragmented genomic DNA and precipitated, followed by DNA purification and sequencing. There are several drawbacks of MeDIP-Seq: 1. Low resolution (150~200 bp) as opposed to the single base resolution; 2. Non-specific interaction due to antibody specificity and selectivity. 3. Bias towards hypermethylated regions.

The Methylation Specific Bisulfite Seq (MSBS) Library Prep Kit (illumina platform) was developed for construction of NGS libraries for methylated CpG sites using bisulfite treated DNA (20 ng – 500 ng) as input. The kit enriches methylated CpG regions, thus significantly reduce the sequencing cost. The kit estimates the whole genome methylation patterns at the single base level since it is based on a bisulfite-seq technology.

It is known that bisulfite treatment of completed NGS libraries causes tremendous damage to the libraries. By using bisulfite treated DNA as input, the kit overcomes the significant library loss due to the bisulfite conversion. The kit contains a mixture of PCR polymerases that have high-fidelity amplification and uracil tolerance which is ideal for bisulfite treated DNA.

Methylation Specific Bisulfite Seq Library Prep Kit Workflow

Three index types are available for the kit:

Non-index (Cat.# 30101): Libraries do not have index.

Index (Cat.# 30102): Each primer contains a unique barcode sequence of 6 bases to identify the individual library. Library multiplexing capacity is up to 48 samples. Index information can be downloaded here.

Unique dual index (Cat.# 30103): The multiplexing of bisulfite sequencing library is up to 96 samples with unique dual indexes. We used a Four-Base Difference Index System to generate indexes that have at least 4 bases different from each other in the 8-base index. The index primers remove NGS errors including index cross-contamination, index hopping, reads mis-assignment etc. Index information can be downloaded here.

Methylation Specific Bisulfite Seq advantages

• • • Enrichment of methylated CpG sites
    • Single-base resolution
    • Low cost for sequencing
    • Fast
      • Total time: 1.5 hours
      • Hands-on time: 10 minutes
    • Simple workflow
    • Bisulfite treated DNA as input: From 20 ng to 500 ng

MSBS Library Prep Kit enriches CpG sites

High methylation regions and low methylation regions in human genome.

High methylation region in human genome.

Low methylation region in human genome.

Sequencing setting: Single-end 35 cycles (Read 1, 35 bases) recommended
To maximize the methylated CpG enrichment, we recommend to sequence the MSBS libraries with single end 35 cycles (read1, 35 bases). This is because the enriched methylated CpG sites are mainly located around the beginning of read 1 sequences. Shorter single end reads tend to have better methylated CpG enrichment.

Bisulfite seq is a well know technology to detect DNA methylation and several technologies such as WGBS, RRBS, MeDIP-Seq, and MSBS are used for whole genome DNA methylation analysis. DNA methylation is important for regulation of cell development, differentiation and gene expression in molecular biology, genetics and epigenetics. Most methylated cytosines are found at CpG sites, and 70-80% of cytosines are methylated. The number of CpG sites in human genome is around 28 million, which is less than 1% of the genome compared with 4.4% expected.